The Mycobacteria are a genus of bacteria which are acid-fast, non-motile, gram-positive rods. The genus comprises several species which include, but are not limited to, Mycobacterium africanum, M. avium, M. bovis, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. microti, M. scrofulaceum, M. paratuberculosis and M. tuberculosis. Certain of these organisms are the causative agents of disease. For the first time since 1953, cases of mycobacterial infections are increasing in the United States. Of particular concern is tuberculosis, the etiological agent of which is M. tuberculosis. M. tuberculosis and other mycobacteria which are closely related to M.tb. (M. bovis, M. africanum, M. tuberculosis BCG and M. microti) are referred to as the TB complex mycobacteria. Many of these new cases of mycobacterial infection are related to the AIDS epidemic, which provides an immune compromised population which is particularly susceptible to infection by Mycobacteria. Mycobacterial infections other than tuberculosis are also increasing as a result of recent increases in the number of immune compromised patients. For example, Mycobacterium avium, Mycobacterium kansasii and other non-tuberculosis mycobacteria are found as opportunistic pathogens in patients infected with HIV as well as in in other immune compromised patients.
In recent years there has also been an increase in the number of clinical isolates of tuberculosis which are resistant to at least one of the antibiotics normally used to treat the disease (e.g., isoniazid, rifampin or streptomycin). Multidrug-resistant tuberculosis strains have emerged in several countries, resulting in a corresponding increase in the number of fatalities in both immunocompetent and immunocompromised individuals. Because M.tb. grows very slowly (doubling time 20-24 hrs.), conventional methods for identifying this organism and determining drug susceptibility require 2-18 weeks. During that time, patients are often treated empirically with antibiotics which may be ineffective, as lack of any treatment allows the patient to remain infectious and puts the patient and patient contacts at risk. Such empirical treatment can also exacerbate the development of drug resistance.
Conventional diagnosis of mycobacterial infections is dependent on acid-fast staining and cultivation of the organism, followed by biochemical and morphological assays to confirm the presence of mycobacteria and identify the species. These procedures are time-consuming, and a typical diagnosis using conventional culture methods can take as long as six weeks. Automated culturing systems such as the BACTEC.TM. system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) can decrease the time for detection of mycobacteria to one to two weeks. Once detected, culturing these slow-growing microorganisms in the presence of antibiotics to determine their drug susceptibility requires several additional weeks. There is still a need to reduce the time required for diagnosing mycobacterial infections and determining antibiotic susceptibility even further in order to allow prompt, informed treatment of M.tb. infections.
The BACTEC TB System provides one means for determining whether or not a positive mycobacterial culture is the result of TB complex mycobacteria or mycobacteria other than tuberculosis (MOTT). This is important information for the initial diagnosis of tuberculosis, and shortens the time required for determining the species present in a positive mycobacterial culture. The BACTEC identification scheme relies on a combination of three tests, namely, morphology on smear, growth characteristics and the NAP (p-nitro-.alpha.-acetylamino-.beta.-hydroxy-propiophenone) TB differentiation test. NAP is an intermediate compound in the synthesis of chloramphenicol which markedly inhibits the growth mycobacteria belonging to the TB complex. MOTT show little or no growth inhibition, and any slight inhibition of growth is usually temporary. The mechanism of action of NAP on TB complex mycobacteria is not known, nor is the reason for its TB complex-specificity. When cultured in the presence of NAP, TB complex organisms show sharply reduced evolution of CO.sub.2, whereas MOTT continue to grow with increasing CO.sub.2 production. The BACTEC TB System measures CO.sub.2 evolution, as a "growth index" (GI) by monitoring production of 14.sub.C -labeled CO.sub.2 in cultures containing 14.sub.C -labeled palmitate. Once a positive culture is obtained, speciation by determining growth (CO.sub.2 production) in the presence of NAP generally requires an additional 4-6 days.
Luciferase is useful as a biological reporter or signal generating molecule because it catalyzes the reaction of luciferin with adenosine triphosphate (ATP), resulting in the production of light. Sensitive light-detection systems are available to detect and measure light (luminescence) generated by this reaction. Luciferase has been used for many years in the standard assay for measuring ATP. The cDNA coding for firefly luciferase (FFluc) has been cloned, which has allowed its use as a direct reporter molecule in a variety of transformed and transfected cells. In mycobacteria, FFluc has been inserted into the genomes of mycobacteriophage and into plasmids as a reporter gene for use in antibiotic susceptibility testing as an in vivo measure of cell viability after exposure to antibiotics. W. R. Jacobs, et al. (1993) Science 260:819 and WO 93/16172. Inhibition of culture growth results in reduced or absent light production from the cloned luciferase gene. This effect has been attributed to reduced amounts of ATP (required for the luciferase reaction) in antibiotic-sensitive cells, which exhibit reduced metabolic activity in the presence of an anti-TB antibiotic.
.beta.-galactosidase is an enzyme which cleaves lactose into glucose and galactose. Other substrates for this enzyme are also known. X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and chlorophenol red-.beta.-D-galactopyranoside are colorimetric substrates for .beta.-galactosidase. Enzymatic cleavage of X-gal produces a reaction product which is blue in color. Enzymatic cleavage of chlorophenol red-.beta.-D-galactopyranoside produces a reaction product which is yellow to red in color. Methyl umbelliferyl-.beta.-D-galactopyranoside is a fluorometric substrate for .beta.-galactosidase which produces a fluorescent signal when enzymatically cleaved. This ability to produce a signal makes .beta.-galactosidase useful as a reporter molecule in conjunction with colorimetric or fluorometric the enzymatic substrates, and these signal generating systems have been used in a variety of biological assays. Like FFluc, the bacterial gene which encodes .beta.-galactosidase (LacZ) has been cloned and used as a reporter gene in recombinant organisms in both inducible and constitutive expression systems.
As used herein, the term "reporter gene" refers to a gene which can be expressed to produce a gene product which directly or through further reaction generates a detectable signal. This signal can be used to detect or identify cells carrying the gene, either on a plasmid or inserted into the genome of the cell. Examples of reporter genes are the gene encoding firefly luciferase (resulting in a luminescent signal upon reaction with luciferin) and the gene encoding .beta.-galactosidase (resulting in a colored or fluorescent signal upon reaction with appropriate enzyme substrates). A mycobacteriophage carrying a reporter gene is referred to herein as a "reporter mycobacteriophage" or "RM." Mycobacteriophage carrying a luciferase reporter gene are referred to as "luciferase reporter mycobacteriphage" or "LRM." Mycobacteriphage carrying a .beta.-galactosidase reporter gene are referred to as ".beta.-galactosidase reporter mycobateriphage" or ".beta.-GRM."
The host range of mycobacteriophage varies greatly, with some capable of infecting only a single species. Certain mycobacteriophage (e.g., TM4 or phAE40) have been characterized as preferentially infecting species of the TB complex, whereas others (e.g., L5) have a very broad range of mycobacterial hosts. A reporter mycobacteriophage constructed in TM4 or phAE40 would therefore be expected to be useful for specific identification of TB complex organisms, as primarily TB complex species should be infected and produce a signal. However, in practice, these mycobacteriophage are not perfectly species-specific, infecting and producing high levels of signal in certain MOTT species as well. This results in false-positives which are unacceptable for clinical detection and identification of TB complex mycobacteria. The present invention not only meets the need for a more rapid method for detection, identification and antibiotic susceptibility testing of TB complex organisms, it solves the problem of identifying false-positives and provides more accurate identification of TB complex organisms using a reporter mycobacteriophage.